Effects of statins on thrombosis development in patients with systemic lupus erythematosus and antiphospholipid antibodies.

The objective of this study is to identify the effects of statins and risk factors for thrombosis in patients with new onset of systemic lupus erythematosus (SLE) with or without antiphospholipid antibodies (aPL). Consecutive patients with SLE without history of thrombotic events were retrospectively enrolled from April 1997 to February 2014. The development of first thrombosis and death caused by thrombosis were defined as the study endpoint. Risk and protective factors for developing thrombosis were analyzed. A total of 152 patients, 80 positive and 72 negative for aPL, were included. In aPL-positive patients, 15 developed arterial ( n = 6) and venous ( n = 9) thrombosis (median follow-up period 69 months). Cox's proportional hazards model showed that older age at SLE onset and IgG-anticardiolipin antibodies (aCL) were statistically significant risks for thrombosis. Statin therapy was identified as a statistically significant protective factor against thrombosis (hazard ratio 0.12, 95% confidence interval 0.01-0.98). In aPL-negative patients (median follow-up period 46 months), seven patients developed thrombosis (five arterial and two venous). No risk factors for thrombosis were found in this group. In aPL-positive patients with SLE, the late disease onset and the presence of IgG-aCL represented additional risk factors for thrombosis. Statin treatment appeared as a protective factor for thrombosis.

CXCR4/fusin is not a species-specific barrier in murine cells for HIV-1 entry.

Since some murine cells expressing human CD4 fail to internalize HIV-1, another block was thought to be located at the level of viral entry in addition to CD4. Recently, CXCR4 was shown to function as a coreceptor for T cell line-tropic HIV-1 entry. Here we demonstrated that cells expressing murine CXCR4 and human CD4 fused with cells expressing the env proteins derived from T cell line-tropic HIV-1 and were infected with T cell line-tropic HIV-1 strains. In contrast, the same cells were not infected with chimeric clones constructed by substitution of monocyte- or macrophage-tropic strain-derived env region or V3 region into T cell line-tropic HIV-1, indicating V3 loop of envelope protein is required for murine CXCR4mediated HIV-1 entry. We conclude that murine CXCR4 is not a species specific barrier to the entry of T cell line-tropic HIV-1.

Clonal expansion of CD8+ cytotoxic T lymphocytes against human T cell lymphotropic virus type I (HTLV-I) genome products in HTLV-I-associated myelopathy/tropical spastic paraparesis patients.

Short-term culture of peripheral blood mononuclear cells (PBMC) derived from patients with human T cell lymphotropic virus type I-associated myelopathy (HAM)/tropical spastic paraparesis resulted in dominance by DR+ activated CD8+ T cells. Variations in the T cell receptor (TCR) V alpha and V beta chains in these cells were analyzed, and in all 10 patients examined, 2-3 V gene families were dominant in both TCR V alpha and V beta. In five patients we examined, cultured lymphocytes contained cytotoxic lymphocytes for p40tax (patients HAM2, 3, 7, and 8) or env protein (patient HAM4) of human T lymphotropic virus type I. In patients HAM2 and HAM8, cultured lymphocytes contained a large proportion of V beta 8+ CD8+ and/or V beta 12+ CD8+ cells. The sequence of V beta 8+ and V beta 12+ cDNA revealed that they were oligoclonal with identical or similar sequences in each patient. Elimination experiments with monoclonal antibodies for TCR V beta 8 and V beta 12 showed that they were CD8+ cytotoxic T lymphocytes (CTL) for p40tax. In addition, flow cytometry and sequencing analysis of uncultured PBMC revealed that in HAM2, V beta 8+ CTL and their precursors account for 7% and V beta 12+ CTL and their precursors account for 18% of total CD8+ cells. This indicates the presence of two markedly expanded clones in vivo. No common dominant TCR V alpha or V beta were observed among 10 HAM patients analyzed.

Variants of vaccinia virus hemagglutinin altered in intracellular transport.

Two classes of revertants were isolated from a vaccinia virus mutant whose hemagglutinins (HAs) accumulate on nuclear envelopes and rough endoplasmic reticulums. The HAs of one of the revertants had the same phenotype as the wild type, i.e., rapid and efficient movement to the cell surface. The HAs of the second class had biphasic transport: rapid export to the cell surface as in the wild type and slow movement to the medial cisternae of the Golgi apparatus. Biochemical and nucleotide sequence analyses showed that the HAs of all the mutants examined that have defects in transport from the rough endoplasmic reticulum to the Golgi apparatus have altered cytoplasmic domains and that the HAs of the second class of revertants lack the whole cytoplasmic domain, while the HAs of the first class of revertants have a wild-type cytoplasmic domain.

A cellular protein which is coprecipitated with HTLV-I rex protein in the presence of the target mRNA.

We examined the cellular protein(s) which can associate with Rex protein of human T cell leukemia virus type I (HTLV-I), using Rex-maltose binding protein (MBP) fusion protein. Immunoprecipitation of RexMBP with anti-MBP antibody revealed that a 24 kD protein (p24) associated with RexMBP only in the presence of Rex-responsive mRNA. The fact that p24 was present in both the nucleus and the cytoplasm is consistent with a role of Rex in the nucleo-cytoplasmic transport of viral mRNAs. P24 did not interact with nonfunctional Rex mutant proteins even if they had RNA binding activity in vitro. These results suggest the possible involvement of p24 in the Rex function through a complex formation with Rex on Rex-responsive mRNA.

Cytotoxic T cell response and expression of the target antigen in HTLV-I infection.

The cytotoxic T cell response of peripheral blood mononuclear cells (PBMC) to in vitro stimulation with human T cell leukemia virus type I (HTLV-I) was compared among HTLV-I-infected individuals with various clinical conditions. Induction of HTLV-I-specific cytotoxic T lymphocytes (CTL) was observed in 57% of asymptomatic HTLV-I carriers, 86% of patients with HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) or other HTLV-I-related inflammatory diseases, and 18% of adult T cell leukemia (ATL) patients. HTLV-I p40tax, one of the major CTL target antigens, has an epitope strongly associated with HLA-A2. HTLV-I p40tax-specific CTL were frequently induced from HLA-A2-positive donors with HTLV-I-related inflammatory diseases regardless of neurological symptoms, but not from all the HLA-A2-positive HTLV-I-infected individuals tested. Leukemic cells of an ATL patient with HLA-A2, whose PBMC did not show an HTLV-I-specific CTL response, could be lyzed by p40tax-specific CTL derived from an HAM/TSP patient. This indicates that i) the presence of a certain HLA presenting CTL epitopes is not the sole determinant of the individual CTL response to HTLV-I, ii) HTLV-I-specific CTL act as potential effectors of anti-tumor surveillance in vivo. The role of HTLV-I-specific CTL, however, may be limited by another in vivo mechanism suppressing the expression of HTLV-I antigens. This suppression, presumably mediated by a plasma factor and commonly observed in HTLV-I-infected individuals, could be one reason for the persistence of HTLV-I-infection.

Prophylactic procedure for inguinal hernia after radical retropubic prostatectomy.

PURPOSE: The incidence of inguinal hernias (IH) after radical retropubic prostatectomy (RRP) has been reported to range from 10 to 50 %, but no prophylaxis for IH has yet been established. We proposed a prophylaxis for IH after RRP. METHODS: A total of 180 patients underwent RRP at our hospital between 2000 and 2011. In January 2008, we started to perform a prophylaxis involving the dissection of the processus vaginalis. This procedure was performed in 73 patients. We then compared the incidence of IH between the patients that did (prophylaxis group) and did not (no prophylaxis group) undergo the prophylaxis. We also studied the risk factors for IH after RRP. RESULTS: In the no prophylaxis group, 25 (23 %) of the 107 patients developed IH, and the IH-free rate at one postoperative year was 86 %. In contrast, only 3 (4.1 %) of the 73 patients in the prophylaxis group developed IH, resulting in IH-free rate of 96 % at one postoperative year (P = 0.0235). Among the patients in the no prophylaxis group, the mean body mass index of the hernia group was significantly lower than that of the no hernia group (P = 0.006). CONCLUSION: Our results suggest that our prophylaxis is useful for preventing IH after RRP.

Intracellular localization and release of eotaxin from normal eosinophils.

Eotaxin is a potent and selective CC chemokine for eosinophils and basophils. We established several monoclonal antibodies (Mabs) allowing the neutralization and measurement of human eotaxin. Using the Mabs as probes, we demonstrated that normal eosinophils contained intracellular granule-associated eotaxin. Quantification of cell-associated eotaxin in different leukocyte subsets revealed that it was principally expressed in eosinophils. Finally, we showed that normal eosinophils released eotaxin upon stimulation with either of two secretagogues, C5a or ionomycin. These findings raise the possibility that eosinophil-derived eotaxin contributes to the local accumulation of eosinophils at the site of inflammation.

Effect of resin use in the post-embedding procedure on immunoelectron microscopy of membranous antigens, with special reference to sensitivity.

To investigate quantitatively the effect of resins on the sensitivity of immunoelectron microscopy of membranous antigen, ultra-thin sections of bovine epithelial tissue embedded in five different kinds of resins [JB-4 (JB4), LR Gold (LRG), Lowicryl K4M (K4M), Quetol 812 (Q812), and Spurr's (Spurr) resin] were labeled specifically with anti-desmosomal glycoprotein I(DGI) antibody followed by protein A-gold (PAG) conjugates. When we compared the labeling intensity expressed as the number of PAG particles per 500-nm length of the desmosomal region along the membrane, three hydrophilic resins (JB4, LRG, and K4M) showed much greater levels of labeling intensity than did epoxy resins (Q812 and Spurr), which had a negative value. The three hydrophilic resins showed only minor differences in their levels of labeling intensity. The intensity obtained with JB4, which was the highest of the three, was further increased by pretreatment of the ultra-thin sections with methyl methacrylate monomer (MM) for 5 min. On the basis of these results, wide applicability of this new technique for membranous antigens, which have been difficult to detect positively by any previously employed techniques, is suggested.

Electron microscopic localization of specific carbohydrate groups in thin sections of tissues embedded in a hydrophilic resin: concanavalin A receptors in mouse spleen.

An improved postembedding method for electron microscopic localization of concanavalin A (Con A) receptors in ultrathin sections is reported. Materials are embedded in a hydrophilic resin, glycol methacrylate copolymerized with glutaraldehyde and urea, using a precisely controlled polymerization schedule that preserves ultrastructural integrity. Ferritin-Con A binds specifically in ultrathin sections of mouse spleen tissue and of Sephadex beads embedded in this resin. Quantitative investigations of procedures for decreasing nonspecific labeling demonstrate that preincubation of sections with bovine serum albumin reduces background to a very low level. This high-resolution method allows macromolecular affinity probes access to receptors in all cellular locations, while maintaining good morphological preservation.

Demonstration of desmosomal antigens by electron microscopy using cryofixed and cryosubstituted skin with silver-enhanced gold probe.

In a previous post-embedding immunogold electron microscopic (EM) studies, localization of various desmosomal antigens was possible at high but not at low magnification. We developed a method for simultaneous demonstration of epidermal desmosomal antigens at both low- and high-power EM magnifications by a method based on cryofixation and acetone cryosubstitution and the use of a 1-nm gold probe with silver enhancement. Ultra-thin sections of Lowicryl K11M were incubated with primary antibodies against desmoplakin, desmocollin, or desmoglein, followed by 1-nm gold-conjugated secondary antibody. Silver enhancement for 12 min provided the ideal labeling size for low-power visualization, whereas silver enhancement for 4-6 min was ideal for high-power EM observation. Each desmosome immunolabeled with the gold probe was clearly demonstrated, even at very low-power magnification. The level of background labeling could be determined easily and the area of interest for high-power observation selected accurately. The fine ultrastructural appearance of desmosomal molecules was precisely demonstrated on high-power observation. This system should be useful for the immunocytochemical study of a variety of desmosomal antigens as well as other molecules of interest.

An antigenic structure of the trans-activator protein encoded by human T-cell leukemia virus type-I (HTLV-I), as defined by a panel of monoclonal antibodies.

In order to study an antigenic structure of the trans-activator protein encoded by human T-cell leukemia virus type-I (HTLV-I), tax1 antigen, we generated and characterized a panel of rat anti-tax1 monoclonal antibodies (MAbs) designated WATM-1, WATM-2, WATM-3, and WATM-4. These MAbs were derived from WKA rats immunized with HTLV-I-transformed (HTLV-I+) syngeneic T cells. Immunoblot assays showed that: (1) All the MAbs reacted with the tax1 antigen in HTLV-I+ cell lines and a recombinant tax1 antigen, PX141 (containing entire tax1 polypeptide); (2) WATM-3 and WATM-4, but not WATM-1 or WATM-2, reacted with a truncated tax1 antigen, XD59 (tax1 amino acids 180-338); (3) None of them reacted with another truncated tax1 antigen, XD128 (tax1 amino acids 1-47 and 286-353); and (4) each of the four MAbs had different reactivity with tax1-related antigens in the range 38-41 kDa expressed in simian cell lines infected with various HTLV-I-related simian retroviruses (STLV-I). None of the MAbs reacted with HTLV-II tax antigen. Human sera containing anti-tax1 antibodies interfered specifically with the antigen-specific binding of all the MAbs. These results suggest that the present rat MAbs are directed against various epitopes on the tax1 antigen. An antigenic structure of the tax1 antigen deduced from reactivity of a panel of anti-tax1 MAbs including the present rat MAbs is discussed.

Functional expression of human mannan-binding proteins (MBPs) in human hepatoma cell lines infected by recombinant vaccinia virus: post-translational modification, molecular assembly, and differentiation of serum and liver MBP.

Human mannan-binding proteins (MBPs) occur in two forms, serum MBP (S-MBP) and liver MBP (L-MBP), both of which are synthesized in the liver from a single form of human MBP mRNA. To investigate further the mechanisms of post-translational modification, molecular assembly and differentiation of S-MBP and L-MBP in vitro, we expressed a full-length human MBP cDNA in three human hepatoma cell lines, using the vaccinia virus expression system. The expression of human MBP cDNA reproduced the native MBP differentiation of S-MBP and L-MBP in human hepatoma cells. The recombinant S-MBP was secreted into the medium, and the recombinant L-MBP retained in the cells. The former had the ability to activate the complement through the classical or lectin pathway but the latter did not. Furthermore, one notable difference between the two MBPs was the degree of oligomerization through interchain disulfide bonds between subunits. In addition, we showed that both S-MBP and L-MBP undergo hydroxylation of lysine and proline residues in collagen-like sequences, and that the hydroxylysine is glycosylated to form glucosylgalactosylhydroxylysine (GluGalHyl) and galactosylhydroxylysine (GalHyl). Hydroxylation was required for S-MBP to be assembled into large complexes, the apparent molecular sizes of which were estimated to be 200-1,300 kDa by SDS-PAGE under non-reducing conditions and gel filtration under non-denaturing conditions. The hydroxylation and subsequent glycosylation and oligomerization were inhibited by alpha,alpha'-dipyridyl, an inhibitor of collagen lysyl and prolyl hydroxylases. These results suggested that newly synthesized lectins undergo post-translational modifications unique to the two forms of MBP, S-MBP, and L-MBP, in human hepatocytes and hepatoma cells, and that the collagen-like domains of the MBPs play an important role in promoting molecular assembly.

Induction of recombinant gene expression in stably transfected cell lines using attenuated vaccinia virus MVA expressing T7 RNA polymerase with a nuclear localisation signal.

There are major drawbacks using vaccinia virus (VV) expressing T7 polymerase for eukaryotic expression. VV is infectious for humans and due to cytosolic replication of Poxviridae, transient transfection of T7 promoter containing plasmids is necessary, which varies in efficiency. Several improvements have been introduced to this system to enhance expression of herpes viral glycoproteins. Stably transfected cell lines were generated with an EBV-based episomal plasmid vector which can be pushed to increasing copy numbers under selective pressure. The avirulent vaccine MVA strain was adopted to generate a safe laboratory vector for inserting the bacteriophage T7 RNA polymerase gene with (+) or without (-) a nuclear localisation signal. Constructs were designed for recombination into the VV haemagglutinin gene as recombinants could not be isolated successfully when inserting into the MVA thymidine kinase locus. Both T7 MVA recombinants induced foreign protein expression in transiently transfected cells but only the T7-/+ MVA induced target protein expression in stably transfected cells. The level of protein expression by this induction mechanism was comparable to, or superior to levels obtained with VV recombinants expressing the gene under control of the VV 11 k IE promoter. The results suggests that the T7+ MVA virus can be used to induce gene expression in stable recombinant cell lines and offers an attractive and safe alternative to other inducible eucaryotic expression systems.

Stress-induced expression of the c-fos proto-oncogene in the hippocampal formation.

To investigate the effects of stress on c-fos mRNA expression, rats were submitted to forced immobilization for 15, 30, 60 or 120 min before sacrifice. In situ hybridization was performed on sections containing the dorsal hippocampus with a 32P-labelled 50-base oligonucleotide probe (10(7)-10(9) cpm/micrograms) complementary to nucleotides 370-319 of rat c-fos. Forced restraint induced a time-dependent increase in c-fos mRNA expression which was most pronounced in the dentate gyrus and CA1-CA3 regions of the hippocampal formation, and which peaked after 30 min of immobilization (72.7 +/- 1.0 vs 24.1 +/- 0.8 cpm/mm2 in unrestrained animals). A positive but weaker signal was also detected in the amygdala, pyriform cortex and other parts of the cerebral cortex and habenulae. These results suggest that the hippocampal formation is activated during stress.

Induction of the c-fos proto-oncogene in the rat pineal gland during stress.

To investigate the effects of stressful stimuli on pineal gland activity, male Wistar albino rats (200-250 g, 2-4 per group) were submitted to 30 min of forced immobilization or to unilateral vibrissotomy 30 min before sacrifice. In situ hybridization was performed with a 35S-labelled 50-base oligonucleotide probe complementary to nucleotides 270-319 of rat c-fos on sections containing the pineal gland. Autoradiograms were quantified using a JAVA microdensitometer. Stressful stimuli induced a significant increase in the expression of c-fos mRNA in the pineal gland (restraint = 144.3 +/- 14.4 cpm/mm2; hemivibrissotomy = 206.7 +/- 29.5 cpm/mm2) as compared to no restraint animals (30.6 +/- 5.1 cpm/mm2), animals displaying tonic-clonic seizures after an ip (64 mg/kg) injection of pentylenetetrazole (34.0 +/- 4.7 cpm/mm2), or competition (70.6 +/- 11.4 cpm/mm2) and RNAase-treated (52.7 +/- 9.1 cpm/mm2) controls. These results raise the possibility that stressful stimuli may interfere with pineal gland function.

Simple deep hypothermia for open-heart surgery.

Two hundred fifty patients more than 2 years of age having correction of congenital heart diseases by simple deep hypothermia alone were investigated in respect to metabolic abnormalities, post-operative complications, intellectual development and postoperative EEGs. LOS in lethal complications was attributed to the difficulty of resuscitation, indicating the application of this method is ideal for patients less than 6 years in age or less than 20 kg in weight. No impairment of intellectual development was observed when compared IQ before the operation and at the time of long term follow-up in serial study, but electroencephalographic assessment indicated that postoperative abnormalities might occur more frequently than previously suspected. Conclusively, it would appear that hypothermic intracardiac surgery is a safe method, provided circulatory arrest time is not allowed to exceed a limited period and the procedure is reasonably performed having a good understanding in the pathophysiology of hypothermia.

Rounded atelectasis associated with silicosis.

Two cases of autopsy-proven, silicosis-associated rounded atelectasis (SARA) are reported. Clinicopathologically, SARA shows a combination of rounded atelectasis and the typical features of silicosis. Pathologically, SARA is characterised by the presence of numerous silicotic nodules deposited throughout the atelectatic lung tissue, which otherwise shows the ordinary features of rounded atelectasis. SARA may contribute to the development of massive fibrosis in silicotic lungs.

Triple lung cancers in a patient with silicosis.

A case of pulmonary silicosis associated with triple lung cancers is presented. Two of the three tumors were unexpected clinically. The discussion deals with the diagnostic difficulty in radiological differentiation between the pneumoconiotic mass lesion and superimposed tumor shadows, as well as the possible causal relationship between the two conditions. From the practical point of view, elderly patients with pneumoconiosis should be carefully monitored for the development of lung cancer.

[A case of multiple liver metastasis from remnant gastric cancer responding to leucovorin.5-FU+UFT therapy].

We report a case of a 67-year-old male patient who experienced multiple liver metastasis 6 months after undergoing an operation for remnant gastric cancer. The histological classification of the cancer in gastric remnant was poorly-differentiated adenocarcinoma. The patient was treated with a low dose of LV.5-FU once a week and oral UFT as an outpatient. As a result, after 3 months of the treatment, CT showed that multiple liver lesions almost disappeared, a condition that lasted about 3 years without relapse. Toxic effects due to this treatment were temporary slight liver disfunction, mild anorexia and stomatitis. This case indicates that the regimen of LV.5-FU+UFT may be effective for multiple liver metastasis from postoperative remnant gastric cancer, enabling the patient to maintain an excellent QOL (quality of life).